During the past year we have continued to examine the role of polyglutamate formation in the cytotoxic action of methotrexate. We have examined various aspects of the formation, retention, and binding of polyglutamates in human breast cancer cell lines. The major findings have been 1) that higher polyglutamates, particularly those with 4 or 5 glutamyl groups, are preferentially retained by human breast cancer cell lines, 2) that these longer polyglutamates have slower rates of dissociation from the enzyme as determined in whole cells, 3) studies with isolate dihydrofolate reductase have shown that methotrexate and its polyglutamates have roughly equivalent dissociation constants, implying that unidentified factors, such as association of DHFR with thymidylate synthetase in intact cells, may account for the slower off rate seen in the whole cell, and 4) methotrexate polyglutamates have additional sites of inhibitory action, including potent inhibition of thymidylate synthetase and AICAR transformylase. We have also identified and described gene amplification in a human small-cell carcinoma line derived from a patient treated with high-dose methotrexate, and have shown that the amplification is unstable in culture, likely because of the failure of the double-minute chromosomes to segregate equally during cell division. The loss of double-minute chromosomes in this cell line during continuous passages in tissue culture was associated with a fall in intracellular DHFR and a return to sensitive state. We have initiated studies on pleiotropic drug resistance in human and murine tumor cell lines. Through continuous exposure to colchicine in tissue culture we have derived 10- to 20-fold resistant cell lines which display cross-resistance to adriamycin and vincristine. In murine cell lines this resistance is reversible with verapamil, while in human breast cancer cell lines the resistance is only minimally reversible. In addition, the human breast lines show a cytotoxic response to verapamil alone. We plan to define the calcium channels present on these cells and will study the relationship of channels to drug resistance. We have also initiated studies attempting to transfer drug resistance through DNA-mediated transfection to sensitive receptor cell lines.